The present invention relates generally to enhancement of the immunological competence of animals, including humans, and more specifically to improved methods for administration of immunologically active nucleopeptide substances isolated from leucocytes of a sensitized donor animal and capable of conferring or mediating immune reactions to antigenic substances. According to the invention substantial therapeutic advances may be achieved in the prevention and/or treatment of disease states in disease host animals which may be naturally immunologically incompetent.
Delayed-type hypersensitivity to tuberculin and chemicals was first transferred passively by intact peritoneal exudative leucocytes in guinea pigs in the 1940s. The inventor and his co-workers, in 1954, reported successful transfer of delayed cutaneous sensitivity to 2,4-dinitrochlorobenzene and tuberculin in guinea pigs with leucocytic extracts from cells disrupted by sonic oscillation [See, e.g., Proc. Soc. Exp. Biol. and Med., 86, 251-253 (1954) and J. Bact., 680-683 (1957)]. Other workers showed similar results in human beings with extracts of blood leucocytes lysed by freezing and thawing. The active component of these extracts was named "transfer factor" and many subsequent studies on the nature and functions of the transfer material have been reported. [See, e.g., Transfer Factor: Basic Properties and Clinical Applications, Asher, et al. (ed.), Academic Press, Inc., New York, New York (1976).]
Available evidence concerning the physical properties of transfer factors generally indicates that they are substantially non-antigenic, dialyzable materials produced in leucocytes and having a molecular weight of from about 500 to 10,000 Daltons -- usually between 1500 to 5000. Transfer factors contain polypeptide and polynucleotide components and have an electrophoretic mobility similar to .alpha. globulins, albumins and pre-albumins. They are unaffected in activity by in vitro treatment with ribonuclease and trypsin as well as deoxyribonuclease. Similar treatment with snake venom phosphodiesterase and certain non-specific proteases (e.g., Pronase, Type VI, Sigma Chemical Co.,) has been observed to interfere with passive transfer capacity. Transfer factors are generally believed to lack sulfur-containing amino acids and solutions give a 260/280 nm optical density ratio which averages from about 0.20 to about 0.80.
Transfer factors are ordinarily isolated directly from disrupted leucocytes of a donor animal, preferably an animal sensitized to a selected antigenic chemical or biological substance including whole, or fractionated extracts of, live or killed infectious microbial materials such as bacteria, viruses, rickettsiae, fungi, protozoa and the like. Collection of leucocytes for isolation of transfer factor materials is frequently accomplished by separation of cells from whole blood, spleen or lymph node tissue or artificially-induced intraperitoneal exudates. Recent developments in processes for securing transfer factors have included isolation of transfer factor fractions from plasma of sensitized donor animals to whom a lympholytic material such as heterologous antilymphocytic serum or globulin has been administered for the purpose of disrupting the structural integrity of leucocytes in vivo.
Transfer factors transmit, upon administration to the general circulatory system of a recipient animal, specific cell-mediated immunity to antigenic substances. The precise mode of operation of transfer factors is as yet not clearly illucidated. It is not known, for example, whether the dialysate materials commonly known as transfer factors are essentially purely nucleopeptides, or whether all or merely a portion of the nucleopeptide component is immunologically active. Similarly unknown is the physiological mechanism by which these non-antigenic materials rapidly interact with the recipient animal's circulatory system constituents to develop delayed-type hypersensitivity reaction to antigens. Transfer factors are presently undergoing extensive human clinical trials with some success in the treatment of certain immuno-deficiency diseases, malignancies, and chronic infections and inflammatory diseases. Therapeutic schemes in effect or under consideration include administration of transfer factors derived from homologous or heterologous species -- either alone or in combination with other chemotherapeutic agents.
Due to the inherent imprecision attending quantification of a transfer factor material of any standardized purity, dosage amounts of transfer factor are expressed in terms of "units" correlated to the quantity of dialysate obtained upon extraction from a given quantity of lymphocytes. Thus a dosage expressed as 1 .times. 10.sup.8 lymphocyte equivalents characterizes the quantity of transfer factor obtained by isolation from 1 .times. 10.sup.8 lymphocytes obtained from a donor animal.
Present modes of administration of transfer factors are exclusively parenteral in nature with human transfer factor usually given either subcutaneously or intradermally. Recent studies with mice have indicated that transfer factor is active when administered intraperitoneally and have suggested that a similar mode of administration be attempted in humans [See, Rifkind, et al., Infection and Immunity: 16, pp. 258-262 (1977)]. To date intravenous administration has been avoided owing to the known substantial risks normally accompanying introduction of proteinaceous substances into the circulatory system. Indeed, potential for anaphylactic-type reactions accompanies even subcutaneous and intradermal administration to such a degree that many investigators have been reluctant to fully explore their therapeutic potential. This is so even though transfer factors isolated by dialysis are generally free of association with higher molecular weight (.about.10,000) polypeptides.
In a like manner oral administration of transfer factors has been avoided due to expectation of degradation, and consequent inactivation, upon exposure to a digestive environment in the alimentary canal. Based on existing knowledge of the nucleopeptide constitution of transfer factors, it might be expected that their immune-donative capacities could withstand degradation by carbohydrases (e.g., amylase in saliva) and simple esterases. Despite the apparent absence of deactivation upon in vitro response to trypsin, however, it has been uniformly believed in the art that transfer factors, like interferon, would not retain activity upon exposure to other proteolytic hydrolytic digestive enzymes in gastrointestinal secretions (e.g., pepsin, chymotrypsin, carboxypeptidase A and B) or in cells of the intestinal mucosa (e.g., the aminopeptidases). These beliefs are substantially supported by a prevailing understanding in the art that proteins, polypeptides and nucleopeptides are not directly transported across the gastro-intestinal membrane, so that even if the transfer factors were able to withstand a digestive environment, they would not be made available to the circulatory system by absorption through gastrointestinal tissue. The exceptions to this "rule" concerning intact absorption of proteinaceous substances are quite minimal. Certain neonatal animals, including humans, transitorily display a potential capacity for absorption of intact protein and immunologically active agents from colostrum and early milk. [See, Wiseman, "Absorption from the Intestine," pp. 65-67, Academic Press, New York, New York (1964) and Schlesinger, et al., The Lancet: September, 1977, pp. 529-532.] Also, systemic anti-inflammatory activity has been ascribed to absorption of trypsin in the human ileum when there delivered in small, enteric-coated doses (See, U.S. Pat. No. 3,004,893).
In sum, transfer factors are presently recognized in the art as substances possessing substantial therapeutic potential but which are as yet incompletely characterized as to immunologically active components or mode of operation, and which are subject to substantial limitations in available modes of safe delivery to animals, including humans.
For purposes of providing a better understanding of background of the invention and particularly the therapeutic uses of transfer factors, the disclosures of commonly owned, co-pending U.S. Application Ser. No. 813,584, filed July 7, 1977, are expressly incorporated by reference herein.